Selection and use of lactic acid bacteria for reducing dental caries and bacteria causing dental caries

ABSTRACT

New strains of  Lactobacillus  that have been selected for their capability of improved reduction the number of  Streptococcus mutans  in the mouth of mammals through inhibiting activity in combination with better binding to the oral mucins and dental plaque, thereby preventing, reducing or treating dental caries, and products derived from said strains, including agents for treatment or prophylaxis of caries for administration to humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.10/869,185 filed Jun. 14, 2004, now U.S. Pat. No. 7,517,681 issued Apr.14, 2009, which is a continuation-in-part of U.S. patent applicationSer. No. 10/353,407 filed Jan. 29, 2003 now U.S. Pat. No. 6,872,565,issued Mar. 29, 2005, the disclosure of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to use of nonpathogenic anti-cariogenic lacticacid bacteria strains, and products and methods using such strains,mutants, metabolites and components thereof for treatment andprophylaxis of dental caries caused by oral bacteria such asStreptococcus mutans, and other caries-causing pathogens.

2. Description of the Related Art

The oral cavity of humans and other mammals contains many differentspecies of bacteria, including a number of different species ofLactobacillus. Caries is a disease caused by bacteria. Already in 1890,Miller in “Chemico-Parasitic Theory” presented the hypothesis thatcaries is caused by oral bacteria producing acids from digestivecarbohydrates, which will dissolve the hydroxyhepatite of the teeth. Itwas later confirmed in gnotobiotic rats, for example, that normal oralbacterial flora, primarily of the mutans streptococci group andsecondarily the lactobacilli group are involved in caries production.These “acidogenic” species resident in the oral cavity are associatedwith the presence and onset of dental caries (Locsche W J, MicrobiologRev., 1986:50:353-380). There are seven bacterial species within thegroup mutans streptococci, where Streptococci mutans (serotype c, e, f)are found in 90% of all human isolates (Linder L., Oral Mikrobiologi1996, ISBN 91-7205-037-3). There is abundant evidence that theinitiation of caries requires a relatively high proportion of S. mutanswithin dental plaque. These bacteria adhere well to the tooth surface,produce higher amounts of acid from sugars than other bacterial types,can survive better than other bacteria in an acid environment, andproduce extracellular polysaccharides from sucrose. When the proportionof S. mutans in plaque is high (in the range 2-10%) a patient is at highrisk for caries. When the proportion is low (less than 0.1%) the patientis at low risk. Because they are more acid tolerant than other bacteria,acid conditions within plaque favor the survival and reproduction ofmutans streptococci.

Two other types of bacteria are also associated with the progression ofcaries through dentin. These are several species of Lactobacillus, andActinomyces viscosus. These bacteria are also highly acidogenic andsurvive well in acid conditions. The involvement of Lactobacillus indental caries has been established (Smith et al., Microbios 105: 77-85,2001). In fact, estimation of the lactobacillus counts in saliva, inaddition to the estimation of mutans streptococci counts, usingdifferent selective media or other techniques, has been used for manyyears as a “caries test” and as a way to attempt to identify groups athigh risk for caries. Thus, Lactobacillus strains, some isolated fromhuman dental plaque, may be highly cariogenic (Fitzgerald et al., J.Dent. Res. 60: 919-926, 1981.

For a bacteria to be a primary pathogen in the formation of dentalcaries it is required that it have a combination of several of therequired characteristics (Linder, 1996): ability to adhere and colonizeon the teeth surface; ability to accumulate in large numbers on alimited surface of the teeth; ability to quickly produce acid fromcarbohydrates found in foods; and ability to continue acid productioneven under low pH in the dental plaque.

Dietary sucrose changes both the thickness and the chemical nature ofplaque. Mutans streptococci and some other plaque bacteria use themonosaccharide components (glucose and fructose) and the energy of thedisaccharide bond of sucrose to assemble extracellular polysaccharides.These increase the thickness of plaque substantially, and also changethe chemical nature of its extracellular space from liquid to gel. Thegel limits movement of some ions. Thick gel-plaque allows thedevelopment of an acid environment against the tooth surface, protectedfrom salivary buffering. Plaque that has not had contact with sucrose isboth thinner and better buffered. A diet with a high proportion ofsucrose therefore increases caries risk. Thicker plaque occurs in pitsand fissures and, in patients with poor oral hygiene, near the gingivalmargin.

Given this concept of the nature of the disease, it is clear thatprevention and treatment of dental caries requires hindering the effectsof S. mutans, for example, through dietary change as means of reducingthe substrate for the bacteria, to reinforce the surface structure ofthe teeth or reduction of the number of S. mutans bacteria. Thus,treatments that have been tried include: efforts at changing themicroflora, using agents such as topical chlorhexidine and topicalfluoride; reducing the amount of dietary sucrose, by dietary change andsubstitution for sweeteners more difficult to metabolize by S. mutans,such as Sorbitol, Aspartan, Xylitol; decreasing the frequency of eating,by dietary choice; adding fluoride, particularly through dailyapplication during tooth brushing; and increasing salivary flow, usingmechanical stimulation during vigorous chewing to enhance flow, bychanging drugs which reduce flow, or by using drugs to enhance flow.Different approaches has been evaluated for preventing dental caries,for example, one composition uses a lytic enzyme produced by abacteriophage specific for Streptococcus mutans (U.S. Pat. No. 6,399,098of Fischetti et al.). Also, a strain of Lactobacillus zeae has beenmodified through genetical engineering to produce an antibody on itssurface to neutralize the detrimental streptococcal bacteria,(Hammarstrom L., July 2002 issue of Nature Biotechnology); however thisapproach with genetically modified organisms faces an unknown safetyapproval situation.

In addition, one strain of Lactobacillus rhamnosus (ATCC 53013, strainGG) has been promoted as a probiotic method of reducing Streptococcussabrinus and mutans streptococci generally (Nase et al., Caries Res. 35:412-420, 2001). Further work showed that use of this strain as a starterin fermenting milk did not influence the titer of antibodies againsthuman cariogenic bacteria that were present in the milk (Wei et al.,Oral Microbio. & Immunol. 17: 9-15, 2002). L. rhamnosus GG differs fromL. reuteri in many ways, including fermentation characteristics andisolation source. Other microorganisms that have been found to haveinhibitory activity against the formation of dental plaque includeEnterococcus, Lactobacillus acidophilus V20, and Lactobacillus lactis1370 (Oh, U.S. Pat. No. 6,036,952). In order to inhibit S. mutans, otherwork has been done using so called “competitive exclusion” concepts. Forexample, L. reuteri strain ATCC 55730 has been shown to inhibit S.mutans (Nikawa H. et al, News release by Hiroshima University Jul. 11,2002). A tablet product which is on the market in Japan called LS1,containing a strain of Lactobacillus salivarius (LS1) (by Frente Ltd.Japan) is claimed to inhibit S. mutans.

Strains of a wide variety of Lactobacillus species, includingLactobacillus reuteri, have been used in probiotic formulations.Lactobacillus reuteri is one of the naturally occurring inhabitants ofthe gastrointestinal tract of animals, and is routinely found in theintestines, and occasionally in the birth channel, breast milk and mouthof healthy animals, including humans. It is known to have antibacterialactivity. See, for example, U.S. Pat. Nos. 5,439,678, 5,458,875,5,534,253, 5,837,238, and 5,849,289. When L. reuteri cells are grownunder anaerobic conditions in the presence of glycerol, they produce theantimicrobial substance known as reuterin (β-hydroxy-propionaldehyde).Other antimicrobial substances beside the traditional organic acids havealso been reported such as “Reutericyclin” (Höltzel, A. et al.Angewandte Chemie International Edition 39, 2766-2768, 2000) and “PCA(pyroglutamic acid)” (Yang, Z. Dissertation, Univ. of Helsinki, March2000), and “Reutericin 6” (Toba T, et al., Lett Appl Microbiol 13:281-6.). Lactobacilli, including L. reuteri, are also well known to havethe ability to inhibit other organisms such as S. mutans through localcompetition of nutrients and other metabolic interactions.

Mucin binding proteins of L. reuteri have been isolated and described.See, for example, U.S. Pat. No. 6,100,388. Lactobacillus strains havebeen reported to adhere to various cell lines and host mucus. This hasbeen speculated to be important for probiotic activity and is derivedfrom the concept of virulence factors in pathogenic bacteria, where vastarrays of such interactions have been discovered during the last decades(Klemm, P. and Schembri, M. A. (2000) Bacterial adhesins: function andstructure. Int. J. Med. Microbiol. 290, 27-35.) It has however not beenso well known that there are important differences between aLactobacillus strains ability to adhere to oral mucin and mucin fromother sources, Some strains are good at adhering to both oral mucin andother mucin, for example gastric mucin, others are only good at adheringto gastric mucin but less good to oral mucin, others does not adherewell to any kind of mucin. It is therefore a part of the selectionmethod of this invention to use oral mucin to find the best strains.

While the possibility of effective antibacterial activity and somebinding characteristics by L. reuteri is known, and S. mutans inhibitingeffects of L. reuteri strain ATCC 55730 and Lactobacillus GG ATCC 53103are also known, and some other lactic acid bacteria have been claimed tobe anti-cariogenic, it was not previously known that substantialdifferences existed between lactobacilli strains in their ability toreduce the number of Streptococci mutans in the oral cavity and therebycaries, nor that such strains could be selected.

It is therefore an object of the invention to provide better strains ofLactobacillus which have been selected for their capability to reducethe number of S. mutans in the mouth through antimicrobial activity incombination with good capabilities of adhering to oral mucin and therebysuccessfully prevent, reduce or treat dental caries. It is a furtherobject of the invention to provide products containing said strains,mutants, metabolites or components thereof, including agents forprophylaxis or treatment of caries associated with S. mutans foradministration to humans.

Other objects and advantages will be more fully apparent from thefollowing disclosure and appended claims.

SUMMARY OF THE INVENTION

The invention herein comprises strains of Lactobacillus that have beenselected for their capability of reducing the number of Streptococcusmutans in the mouth of mammals through inhibiting activity, includingmutants, metabolites and components thereof, in combination with goodbinding of the Lactobacillus to oral mucin and dental plaque forpreventing, reducing or treating dental caries, and products derivedfrom said strains, including agents for treatment or prophylaxis ofcaries for administration to humans.

Other objects and features of the inventions will be more fully apparentfrom the following disclosure and appended claims.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS THEREOF

The present invention provides a product, for inhibiting the growth andactivity of dental caries bacteria, comprising cells or metabolites orcomponents of at least one selected strain of Lactobacillus with goodantimicrobial activity against Streptococcus mutans and good bindingcharacteristics to oral mucin and thereby prevent, reduce or treatdental caries. Such strains includes L. reuteri CF2-7F (ATCC PTA-4965),L. reuteri MF2-3 (ATCC PTA-4964) and especially L. reuteri FJ1“Prodentis” (ATCC PTA-5289) and L. reuteri FJ3 (ATCC PTA-5290). Thesestrains are available to the public under the Budapest Treaty at theAmerican Type Culture Collection (Rockville, Md.) the last two havingbeen deposited there on Jun. 25, 2003. All restrictions uponavailability to the public will be irrevocably removed upon granting ofthe patent.

In the selection method used herein, the inhibiting effects ofStreptococci mutans are examined by traditional microbiological methodswith the bacterial cells as well as separately analyzing inhibition bysecreted metabolites and components in the supernatant of the grown teststrains, measured by a method of determining the ATP (adenosinetriphosphate) level of Streptococcus mutans which correlates well withthe total cell volume of viable S. mutans cells. (ref. Nikawa. H. et al.Journal of Dentistery, Vol 26, No 1, pp. 31-37, 1998). The adhesioncapabilities are measured using oral mucin coated in microtiter wells(ref. Jonsson et al. 2001 FEMS Microbiol. lett. 204: 19-22). The reasonfor also testing inhibition and adhesion by secreted metabolites andcell components is that the present invention also could be used withnon-viable cells or parts of dead cells fulfilling the selectioncriteria.

The details of this will be more clearly understood from the Examples.

The product of the invention can be any product for placement in themouth as a preventative or treatment for dental caries, or fornutritional or breath purpose, such as food products, dental treatmentproducts such as mouthwashes or other specified health products, chewinggum, lozenges and the like. Food products lending themselvesparticularly to use in the invention include milk-containing productssuch as yogurt, and also juices, drinks and the like. The dentaltreatment products that may be used in the invention includetoothpastes, liquid tooth cleansers, mouthwashes, anti-halitosisproducts, and the like.

The concentration of selected Lactobacillus cells or metabolites orcomponents thereof, needed for effectiveness of a product of theinvention depends on the type of food and the amount of food to beingested (or the time of use in the mouth of a non-food dental treatmentproduct), but it is usually preferable to have equivalent of about10⁵-10⁸ CFU (colony-forming units) or more per daily intake of aproduct. Amounts up to about 10¹⁰⁻10¹¹ CFU are possible and can be usedto increase efficacy without adversely affecting the product'sorganoleptic characteristics (its flavor or smell). When the product isyogurt or other lactic acid fermentation product, the lactic acidfermentation strain(s) used to produce the product would preferably bestandard cultures for this particular purpose, and the anti-cariogenicbacteria, or metabolites or components thereof, of the invention may beadded either before or after the fermentation of the product at a levelequivalent of about 10⁶-10⁸ CFU per daily serving of yogurt or more asdiscussed above.

Preferably the product of the invention does not contain otherantibacterial components, at least none that inhibit or kill selectedLactobacillus strain(s), or metabolites or components thereof, orinterfere with its anti-cariogenic activity.

The strain(s) of Lactobacillus, or metabolites or components thereof,can be an additive mixed into the ingredients or kneaded into or coatedon the product by means known in the art for formulation of products ofthat type. When using cells and if preparation of the selected food orother product of the invention requires a heating step, theLactobacillus strain(s) should be added after the heating. Once theselected Lactobacillus cells are in the product, it is preferred not toheat the product to 60-70 degrees C. or above for a longer period oftime.

The features of the present invention will be more clearly understood byreference to the following examples, which are not to be construed aslimiting the invention.

EXAMPLE 1 Method of Selection of Strains

The selection of the Lactobacillus strains to be used according to thisinvention can be done in the following three step manner:

a) Evaluation of Inhibiting Effect of S. Mutans by Cells ofLactobacillus Strains

An example of a strain to use to measure the inhibitory effect isStreptococcus mutans, ATCC25175 (available from The American TypeCulture Collection, Manassas, Va., USA). The isolate is grown intrypticase soy broth (Difco, Detroit, USA) supplemented with 0.5% yeastextract (Difco) (TSBY). The cells are harvested during the exponentialgrowth phase by centrifugation at 1000×g, washed twice with PBS andresuspended in the same buffer. The cell suspensions are subjected to alow-intensity ultrasonic device to disperse bacterial aggregates.

The test Lactobacillus strain is grown in MRS broth (Difco), andharvested during the exponential growth phase by centrifugation at1000×g, washed twice with phosphate buffered saline (PBS; pH 6.8) andre-suspended in the same buffer.

The optical densities of the bacterial suspensions are measured in a 1.0ml cuvette with a 1 cm light path, and the suspensions are adjusted to afinal concentration of 1.0×10⁸ CFU (colony forming unit)/ml.

The inhibitory assay is conducted as follows, the suspension of S.mutans and the suspension of Lactobacillus are mixed in the ratios of100-0, 75-25, 50-50 and 25-75 in sterile centrifugation tube (totalvolume 100 μL), added the BHI broth up to 10 ml, vortex mixed for tenseconds and incubated for 90 min at 37° C. with gentle shaking. As acontrol, the suspension of S. mutans is mixed with an equal volume ofPBS in the control tubes (free of Lactobacillus). Afterwards eachsuspension is washed by centrifugation at 1000×g, washed twice with PBS,and plated on MS agar to determine the CFU count of S. mutans. The %survival of S. mutans is obtained from following formula.

${\%\mspace{20mu}{survival}\mspace{14mu}{of}\mspace{14mu}{S.\;{mutans}}} = \frac{{CFU}\mspace{14mu}{of}\mspace{14mu}{S.\;{mutans}}\mspace{14mu}{incubated}\mspace{14mu}{with}\mspace{14mu}{Lactobacillus}\mspace{14mu} \times 100}{{CFU}\mspace{14mu}{of}\mspace{14mu}{S.\mspace{11mu}{mutans}}\mspace{14mu}{incubated}\mspace{14mu}{with}\mspace{14mu}{PBS}}$

The assay should be carried out with minimum triplicate samples. All thenumerical data obtained should be statistically analyzed.

b) Evaluation of Inhibiting Effect of S. Mutans by Metabolites orComponents of Lactobacillus Strains

Also used here as an example of a strain to use to measure theinhibitory effect is Streptococcus mutans, ATCC25175 (available from TheAmerican Type Culture Collection, Manassas, Va., USA). The isolate isgrown in trypticase soy broth (Difco, Detroit, USA) supplemented with0.5% yeast extract (Difco) (TSBY). The cells are harvested during theexponential growth phase by centrifugation at 1000×g, washed twice withPBS and resuspended in the same buffer. The cell suspensions aresubjected to a low-intensity ultrasonic device to disperse bacterialaggregates and adjusted to final concentration of 10⁸ CFU/mL.

The test Lactobacillus strain is grown in MRS broth (Difco), harvestedduring the exponential growth phase by centrifugation at 1000×g, washedtwice with phosphate buffered saline (PBS; pH 6.8) and re-suspended inthe same buffer.

The optical densities of the bacterial suspensions are measured in a 1.0ml cuvette with a 1 cm light path, and the suspensions is adjusted to afinal concentration of 1.0×10⁸ CFU/ml. 100 μL of the lactobacillisuspension was added to 2.0 mL of MRS broth, and incubated at 37° C. for48 h with reciprocal shaking (120 rpm). After the incubation, thelactobacillus cells where removed by centrifugation, and the resultingsupernatant was filtered (pore size 0.25 μm).

The inhibitory assay was conducted as follows. 100 μL of the suspensionof S. mutans was added to 1.0 mL of TSBY and 1.0 mL of the supernatantsof each tested strain of lactobacilli was mixed, and incubated for 24 hin 37° C. with reciprocal shaking (120 rpm). As a control the suspensionof S. mutans was mixed with equal volume of MRS in the control tubes(free of supernatants from lactobacilli). Afterwards each suspension waswashed by centrifugation at 1000×g, washed twice with PBS and the amountof adenosine triphosphate of grown S. mutans was determined using themethod described in; Nikawa. H. et al. Journal of Dentistery, Vol 26, No1, pp. 31-37, 1998.

c) Evaluation of Adhesion Capabilities to Oral Mucin of LactobacillusStrains

The Lactobacillus strains to be tested are collected. The bacteria aregrown at 37° C. in MRS broth (Difco) for 16 h. Plates are incubated inanaerobic jars under CO₂+N₂ atmosphere (GasPak System, BBL, BectonDickinson Microbiology Systems, Cockeysville, Md., USA).

Oral mucus as human saliva are collected, centrifuged, sterile filteredand coated into microtiter wells as described. The mucus are collectedin 200 ml ice-cold phosphate-buffered saline (PBS) (8.0 g NaCl, 0.2 gKCl, 1.44 g Na₂HPO₄.2H₂O and 0.2 g KH₂PO₄ per 1000 ml of dH₂O) andsupplemented with 0.05% Tween 20 (PBST). The resulting suspension iscentrifuged first at 11000 g for 10 min and then at 26000 g for 15 minin order to remove cells and particulate matter. As an alternative mucingastric mucin is (Sigma, M1778) used. The crude mucus preparation isstored at 20° C. The mucus material is diluted to approximately 100 μgml-1 in 50 mM Na2CO3 buffer, pH 9, 7 and incubated overnight inmicrotiter wells (Greiner) (150 μl per well) at 4° C. with slowrotation. The wells are blocked with PBS with 1% Tween 20 for 1 h andthereafter washed with PBST. Wells coated with BSA are used as controls.

The strains to be tested are grown as per above, washed once inphosphate-buffered saline (PBS) (pH 7.3) supplemented with 0.05% Tween20 (PBST) and diluted to OD₆₀₀ 0.5 in the same buffer. One hundredmicroliters bacterial suspension is added to each well and incubatedover night at 4° C. The wells are washed 4 times with PBST and bindingexamined with an inverted microscope. The buffer is poured off and,after the wells had dried, the binding is measured over the wholesurface of the well in a BioRad Gel Doc 2000 instrument (BioRadLaboratories, Herkules, Calif., USA). All measurements are done intriplicate.

The Lactobacillus strains showing best results in both inhibiting of S.mutans using Lactobacillus cells and inhibiting S. mutans usingmetabolites and components of said Lactobacillus as well as best resultsin adhesion to oral mucin according to the assays, are selected.

EXAMPLE 2 Selection of Strains

1. L. reuteri SD2112 (ATCC 55730)

2. L. reuteri DSM 20016 (DSM 20016)

3. L. reuteri MM2-3 (ATCC PTA-4659)

4. L. reuteri CF2-7F (ATCC PTA-4965)

5. L. reuteri MF2-3 (ATCC PTA-4964)

6. L. reuteri MF14-C (Culture collection of Biogaia AB, Raleigh N.C.,USA)

7. L. reuteri MF52-1F (Culture collection of Biogaia AB, Raleigh N.C.,USA)

8. L. reuteri MM7 (Culture collection of Biogaia AB, Raleigh N.C., USA)

9. L. reuteri FJ1, “Prodentis” (ATCC PTA-5289)

10. L. reuteri FJ3 (ATCC PTA-5290).

11. L. salivarius LS1 (isolated from the LS1 tablet by Frente Ltd.Japan)

12. L. rhamnosus GG (ATCC 53103)

In this study the above listed Lactobacillus strains are chosen to beevaluated using the selection criteria of inhibition of S. mutans andadhesion to oral mucin, the methods set forth in Example 1 are used. TheLactobacillus strains showing best results in both inhibiting of S.mutans using Lactobacillus cells and inhibiting S. mutans usingmetabolites and components of said Lactobacillus as well as best resultsin adhesion to oral mucin according to the assays, are selected. Resultsof inhibition are shown in Table 1, and results of adhesion are shown inTable 2.

TABLE 1 Inhibition of S. mutans of Lactobacillus strains as cells aswell as with supernatants of said Lactobacillus according to thedescribed assays. CFU/g CFU/g CFU/g pmol/L survival survival survivalATP amount of S. mutans S. mutans S. mutans S. mutans in SelectionStrain ratio 10:1 ratio 3:1 ratio 1:1 supern. assay to next step L.reuteri SD2112 2.0E+08 8.0E+07 6.0E+07 94644.9 — L. reuteri DSM 1.0E+082.0E+07 7.0E+06 479.1 S 20016 L. reuteri MM2-3 1.0E+08 7.0E+07 5.0E+0735125.3 — L. reuteri CF2-7F 1.0E+08 1.0E+07 7.0E+05 438.6 S L. reuteriMF2-3 2.0E+08 2.0E+07 4.0E+06 33158.8 S L. reuteri MF14-C 9.0E+078.0E+07 7.0E+07 29644.3 — L. reuteri MF52-1F 1.0E+08 8.0E+07 7.0E+073120.7 — L. reuteri MM7 2.0E+08 8.0E+07 7.0E+07 100110.0 — L. reuteriFJ1 1.0E+08 4.0E+06 7.0E+04 3374.3 S L. reuteri FJ3 1.0E+08 1.0E+074.0E+05 11364.1 S L. salivarius LS1 1.0E+08 8.0E+08 7.0E+09 502.8 — L.rhamnosus GG 2.0E+08 8.0E+07 7.0E+07 16113.2 — (S = selected)

TABLE 2 Adhesion of Lactobacillus strains according to the describedassays. Selec- Se- ODxmm² ODxmm² tion lection Final Gastric Oral thisearlier selec- Strain mucin mucin step step tion L. reuteri SD2112 0.390.78 — — — L. reuteri SDSM 9.42 7.85 S S FS 20016 L. reuteri SMM2-3 11.63.55 S — — L. reuteri SCF2-7F 11.73 5.77 S S FS L. reuteri SMF2-3 6.042.82 S S FS L. reuteri SMF14-C 0.2 0.17 — — — L. reuteri SMF52-1F 0.941.45 — — — L. reuteri SMM7 0.39 2.14 — — — L. reuteri SFJ1 7.51 2.77 S SFS L. reuteri SFJ3 7.11 5.9 S S FS L. salivarius SLS1 0.19 1.15 — — — L.rhamnosus SGG 0.5 0.33 — — — (S = selected) (FS = Finally selected)

EXAMPLE 3 Manufacturing of Products Containing Selected Strain

In this example, L. reuteri FJ1 “Prodentis” (ATCC PTA-5289), is selectedbased on good growth characteristics in general and favorable results inthe earlier mentioned selection in Example 2 using the methods above forS. mutans inhibition and mucin binding, in order to add the strain to achewing gum. The L. reuteri strain is grown and lyophilized, usingstandard methods for growing Lactobacillus in the industry.

The steps of an example of a manufacturing process of chewing gumcontaining the selected strain follow, with it being understood thatexcipients, fillers, flavors, encapsulators, lubricants, anticakingagents, sweeteners and other components of chewing gum products as areknown in the art, may be used without affecting the efficacy of theproduct:

1 Melting. Melt Softisan 154 (SASOL GMBH, Bad Homburg, Germany) in avessel and heat it to 70° C. to assure complete disruption of thecrystalline structure. Then cool it down to 52-55° C. (just above itshardening point).

2 Granulation. Transfer Lactobacillus reuteri freeze-dried powder to aDiosna high-shear mixer/granulator, or equivalent. Add slowly duringapproximately 1 minute the melted Softisan 154 to the Lactobacillusreuteri powder. No additional massing time is required. Use chopperduring the addition.3 Wet-sieving. Immediately after the granulation, pass the granulesthrough a 1-mm sieving net by using a Tornado mill. The sieved granulateis packed in alupouches made out of PVC-coated aluminum foil, sealedwith a heatsealer to form a pouch, together with desiccant pouch, andstored refrigerated until mixing. The granulated batch is divided fortwo tablet batches.4 Mixing. Mix all the ingredients in a mixer, to a homogenous blend.5 Compression. Transfer the final blend to the hopper of a rotary tabletpress and compress tablets with a total weight of 765 mg, in a Kiliancompresser.6 Bulk packaging. The chewing gums are packed in alu-bags together witha drying pouch of molecular sieve. The alu-pouch is put in a plasticbucket and stored in a cool place at least one week, before finalpackage.

In-process controls, as is standard in the industry, are shown in thefollowing Table 3:

TABLE 3 Test IPC Method Limit 1 Appearance Clear, homogenous solutionVisually 2 Temperature 52-55° C. Thermometer 3 L. reuteri assay CM003 4Appearance Cream colored with blue Visually spots, convex tablets plainon both sides. Uniformity of mass 765 mg ± 5% Ph. Eur.

In the example herein, the selected L. reuteri culture is then added asabove at a level of 10⁷ CFU/gram of product, and the chewing gum used byhumans as a way to prevent caries. The use of SOFTISAN™, a hydrogenatedpalm oil, enables the Lactobacillus cells to be encapsulated in fat andenvironmentally protected, and is another particularly unique aspect ofthe preferred embodiment of the invention herein.

As stated above, the product of the invention may be in forms other thanchewing gum, and standard methods of preparing the underling underlyingproduct as are known in the art are beneficially used to prepare theproduct of the invention including the selected L. reuteri culture.

While certain representative embodiments have been set forth herein,those skilled in the art will readily appreciate that modifications canbe made without departing from the spirit or scope of the invention.

1. A method of inhibiting growth of dental caries bacteria comprisingorally administering to mammals a product comprising cells of abiologically pure culture of at least one strain of Lactobacillusreuteri selected by a method comprising the steps of: a) selecting andgrowing a strain of Lactobacillus reuteri; b) evaluating the inhibitoryactivity against Streptococcus mutans of said Lactobacillus reuteristrains or metabolites or components obtained from a culture of saidstrain; c) evaluating the adhesion capabilities to oral mucins of saidLactobacillus reuteri strains or metabolites or components obtained fromthe culture of said strain; and d) selecting the strain of Lactobacillusreuteri exhibiting a good inhibitory activity against Streptococcusmutans in combination with good binding to oral mucins.
 2. The methodaccording to claim 1, wherein the product is a food product.
 3. Themethod according to claim 2, wherein the food product is selected fromthe group consisting of yogurt, jelly, pudding, chewing gum, candy,lozenge, chocolate, biscuit, cookie, cheese, juice and tea.
 4. Themethod according to claim 2, wherein the food product is amilk-containing product.
 5. The method according to claim 4, wherein thefood product is yogurt.
 6. The method according to claim 1, wherein theproduct is a dental treatment product.
 7. The method according to claim6, wherein the product is a mouthwash.
 8. The method according to claim1, wherein the cells of the biologically pure culture of at least onestrain of Lactobacillus reuteri are encapsulated in the product.
 9. Themethod according to claim 8, wherein the cells of the biologically pureculture of at least one strain of Lactobacillus reuteri are encapsulatedin a hydrogenated palm oil in the product.
 10. A method of inhibitinggrowth of dental caries bacteria comprising orally administering tomammals a product comprising cells of a biologically pure culture of atleast one strain of Lactobacillus reuteri selected by a methodcomprising the steps of: a) selecting and growing a strain ofLactobacillus reuteri; b) evaluating the inhibitory activity againstStreptococcus mutans of said Lactobacillus reuteri strains ormetabolites or components obtained from a culture of said strain; c)evaluating the adhesion capabilities to oral mucins of saidLactobacillus reuteri strains or metabolites or components obtained fromthe culture of said strain; and d) selecting the strain of Lactobacillusreuteri exhibiting a good inhibitory activity against Streptococcusmutans in combination with good binding to oral mucins, wherein the atleast one strain is selected from the group consisting of: Lactobacillusreuteri strain FJ1 (ATCC PTA-5289), and Lactobacillus reuteri strain FJ3(ATCC PTA-5290).